Ion exchange chromatography is one of the most widely used methods for separating, identifying and/or quantifying amounts of proteins and/or peptides in a mixture or solution. The technique primarily exploits differences in the sign and magnitude of the net electric charges of peptides and/or proteins at a given pH. These values are predictable from the associated pKa value and/or titration curve. See Principles of Biochemistry, Lehninger et al. (New York, 1997, p. 122).
Commonly, a chromatographic column consists of a tube at least partially filled with particles of a synthetic resin containing fixed charged groups. Those with fixed anion groups are called cation-exchange resins. Those with fixed cation groups are called anion-exchange resins. Separation of peptides and/or proteins can occur by gradually changing the pH and/or salt concentration of a solution being run through a column.
An ion exchangers operation is dependent upon certain properties of the system. Each property has an effect on the efficiency and productivity of an ion exchanger. Properties of an ion exchange column that affect operation include, but are not limited to:
Density: The density of resin has an affect upon how the system performs. Properties of resin should be understood. For example, the density of a dry, water free resin is generally smaller for anion exchangers than cation exchangers. The density of water swollen resin depends on the type counter ion, swelling capacity and on the degree of crosslinking, besides the density of dry resin. Furthermore, it should be noted that bulk density is different than the density of the swollen resin. These densities are important because operation is typically dependent upon the resins.
Mechanical resistance: the mechanical resistance is a variable that is studied for ion exchangers. The mechanical resistance is found to vary with structure of the system. It should be noted that air dried resin is destroyed by certain friction. This needs to be thought of in design stages.
The particle size, is a major part of the fluid flow and effectiveness of separation processes. For example, condensation type resins are generally broken granules. On the contrary, polymerization-type resins are small beads that are uniformly packed. To measure the particle size a mesh is used to differentiate larger particles. For separations such as chromatography, particle size can be extremely important to efficiency, especially in regards to resolution of different species. Particle size also largely determines the fluid resistance of an ion exchange column. This can be the key to success of an industrial operation.
The total capacity is a measurement tool used to rate an ion exchanger. The total capacity is the amount of exchangeable ions of unit weight of resin, commonly expressed as ligand density. The determination of such factor can be done by acid-base titration. Another capacity measurement is salt splitting. This is the amount of sodium ions absorbed by the cation exchanger in the hydrogen form from a sodium chloride solution or hydrogen released by unit weight or unit volume. For an anion exchanger the amount of base liberated from a salt by unit weight or unit volume of the hydroxyl-form anion-exchange resin. Dissociation constants of active groups of the resin are a major part of the salt splitting capacity. Further, noted is the rest capacity which consists of the difference in mono-functional strongly acidic or basic resin of splitting capacity. Also, the apparent capacity can be defined as the effects of multivalent ions on an ion exchanger. Further, the break-through capacity depends on the pH, particle size, column size and flow-rate. Knowing and understanding the capacities allows for proper design of the system properties.
The porosity of a system controls much of the capacity of the exchanger. The surface active groups and capillary groups take part in the characteristics of a ion exchanger. The pores of ion exchangers are typically of variable size even for the same resin product. The determination of porosity can be done by means of solution containing ions of known size and similarity by using capacity measurements. Also, the same measurement can be done by the use of vapor pressures. Although these methods only measure mean particle size, it results in useful knowledge. In addition to the above, it should be noted that the degree of crosslinking affects mean pore size.
Throughput: Throughput is an important aspect when considering operational costs and efficiency. Knowing the effects of controlling the flow is desirable also. For example, it is accepted in the art that natural zeolite exchangers operate slower and an ion exchanger of larger pores quicker. As well, a cation exchanger is also known in the art to equilibrate more quickly. The diffusivity is a controlling factor in determining the operating rate. In addition, the rate depends on diffusivity constants of active groups of the resins. However, other influencing factors include, but are not limited to, temperature, solution viscosity, resin density, particle size and distribution, and degree of crosslinking in the resin.
There have been numerous ion exchange chromatography systems established. Each has certain benefits and each has resulted in certain limitations. Ion exchange chromatographic columns are a commonly used step for protein purification. However, the use of ion exchange chromatography is often limited by solutions containing excessive amounts of competing ions for the binding sites. For example, the salt concentration in the loading feed stream from harvested cell culture broth frequently prevents the protein of interest from binding to the resin. This application of an ion exchange column demonstrates the purification of a target molecule in the presence of high salt, in particular product from harvested cell culture broth.
Examples from the prior art include an article by P. Gagnon et al. in the Journal of Chromatography in 1996, vol. 743 (1), pp. 51-55, that disclosed the addition of polyethylene glycol (PEG) to the mobile phase of a column (the phase traveling through the column). The article disclosed that the addition of the PEG altered the retention behavior of proteins and produced unique selectivity in ion-exchange chromatography. However, the article further disclosed that the secondary effects of increased viscosity from addition of PEG severely limited the preparative potential for application of this technique, i.e. elevated viscosity reduced flow-rate. Moreover, the increased viscosity was disclosed as severely depressing the dynamic binding capacity for small proteins, even though the dynamic binding capacity appeared to be maintained or slightly increased for large ones. Likewise, the article disclosed that the PEG addition substantially increased peak width.
Accordingly, the Gagnon article does not disclose a chromatographic system wherein an addition of PEG increases dynamic binding capacity for proteins. Particularly, this article does not disclose a system wherein an addition of PEG increases the dynamic binding capacity for systems with small proteins while reducing process time.
An article authored by K. Milby et al., in the Journal of Chromatography (1989, vol. 482 (1), pp. 133-44), further discloses the addition of PEG to an eluent that increased retention time on tested proteins. However, significant operation pressure increase was observed because of the viscosity of the mobile phase. Importantly, the Milby article does not disclose the addition of PEG in a load feed stream of an ion exchange column.
An article authored by Y. Papanikolau et al., in Protein Engineering (1997, vol. 10 (8), pp. 847-850), makes theoretical assumptions of PEG addition in protein solutions. However, the article failed to state any experimental results or present any other data that would lead one of ordinary skill in the art to add PEG to a load stream in an ion exchange column
An article authored by Feng et al., in Biotechnology Techniques (1998, vol. 12 (4), pp. 289-293), discloses the purification of human tumor necrosis factor-α using anion exchange chromatography. The authors observed that the column binding capacity increased by adding PEG to the buffer and feed solution. The optimum concentration was 1% with PEG 200. However, the Feng article did not disclose the addition of PEG to a protein solution for loading in high salt concentrations.
An article authored by H. Bioerlinq in Vox Sang (1985, vol. (49), pp. 240-243), discloses the isolation of human albumin using a PEG step precipitation. The author observed that all the proteins in the plasma preparation were bound to the DEAE Sepharose gel in the presence of the high salt concentration and with a much higher binding capacity than without PEG. However, the Bioerling article utilizes a PEG concentration of 10% w/w. This severely restricts the application in terms of both solution viscosity and also losses due to precipitation.
U.S. Pat. No. 5,151,358 discloses the recovery and purification of chymosin using a PEG liquid-liquid two-phase separation. The PEG rich product containing profile was than loaded to an ion exchange column. There was no data reported. Moreover the two-phase extraction appeared to be in low salt concentrations.
Accordingly the art field is in search of a process whereby an ion exchange column may be utilized for the purification of product in the presence of high salt, in particular from harvested cell culture broth, thereby allowing a product of interest to bind to a resin.